Analyte detection by competitive inhibition of ion channel gating

ABSTRACT

Membrane for use in the detection of an analyte having a closely packed array of self-assembling amphiphilic molecules and a plurality of ionophores. First and second ligands are attached to an end of the ionophores adjacent the surface of the membrane. The membrane is such that the binding of the first ligand to its specific binding partner prevents the flow of ions across the membrane via the ionophores. In addition, the binding of the second ligand to its specific binding partner prevents the binding of the first ligand to its specific binding partner.

The present invention relates to a membrane incorporating ionophores theconductance of which may be gated.

BACKGROUND OF THE INVENTION

The concept of membranes incorporating ionophores, the conductance ofwhich are gated and the use of such membrane in biosensors is disclosedin International patent application Nos WO89/01159, WO90/08783,PCT/AU89/00352, PCT/AU92/00132 and PCT/AU93/00509. The disclosure ofeach of these documents is incorporated herein by reference.

The present invention stems from an observation made by the presentinventor during the work conducted and disclosed in Internationalapplication No WO90/08783. In this earlier application it was shown thatthe conduction of biotinylated gramicidin ion channels in a lipidmembrane is greatly reduced by the binding of streptavidin by thebiotins attached to the gramicidin, and that the changing conduction inthis system is directly related to the quantity of streptavidin bound tothe membrane ion channels. From this observation the present inventorhas developed a general mechanism for analyte detection using a membraneincorporating ionophores.

DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect the present invention consists in amembrane for use in the detection of an analyte, the membrane comprisinga closely packed array of self-assembling amphiphilic molecules, aplurality of ionophores and a first and a second ligand attached to anend of the ionophore adjacent the surface of the membrane, characterisedin that the binding of the first ligand to its binding partner preventsthe flow of ions across the membrane via the ionophores and in thatbinding of the second ligand to its binding partner prevents the bindingof the first ligand to its binding partner.

In a preferred embodiment of the present invention the first ligand isbiotin, the binding partner of which is streptavidin.

In a further preferred embodiment of the present invention theionophores are gramicidin or gramicidin analogues.

Typically, the second ligand is the analyte which the membrane is to beused for detecting. It is, however, possible that the second ligand mayvary from ionophore to ionophore. In this manner the membrane could beused to detect the presence of more than one analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the nature of the present invention may be more clearlyunderstood the operation of the membrane of the present invention willbe described with reference to the following schematic representationsof the operation of the gating mechanism, in which

FIG. 1 is a schematic representation of a modified gramicidin ionchannel with two ligands attached to the channel;

FIG. 2 is a schematic representation of the effect of addinganti-analyte antibody to the arrangement shown in FIG. 1;

FIG. 3 is a schematic representation of the use of the system shown inFIG. 1;

FIG. 4 is a graph of final/initial impedance against concentration of2,4-dinitroanaline; and

FIG. 5 is a representation of gramicidin B.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a modified gramicidin ion channel (10) with two ligands (12and 14) attached to the C terminus of the channel (10). One ligand (12)is biotin and is capable of binding streptavidin (16) so as to reducethe gramicidin (10) conductance. The second ligand (14), connected inclose proximity to the biotin ligand (12), is the analyte of interest,an epitopic portion of the analyte, a structural analogue of either ofthe above or any ligand capable of competitively binding with anantibody raised against the analyte. In the absence of any speciesbinding to the second ligand (14), binding of streptavidin (16) to thebiotin ligand (12) effects a gating of the channel (10).

FIG. 2 demonstrates the effect of adding an anti-analyte antibody (18)to the system described above. Attachment of the antibody (18) to thesecond ligand (14), while not of itself effecting the conductance ofchannel (10), sterically precludes binding of streptavidin (16) to thebiotin ligand (12). As many channels (10) as have bound antibody (18)are, therefore, unable to be gated.

The use of this system to measure analyte concentrations is illustratedin FIG. 3. In the presence of free analyte (20) in the sample solution,anti-analyte antibody (18) antigen binding sites will be occupied by thesolution analyte (20), in proportion to the analyte concentration. Theseantibodies (18) will be prevented from binding to the second ligand (14)on the ion channel (10), leaving a fraction of ion channels (10)available for gating by streptavidin (16) in proportion to the amount ofanalyte (20) present in the sample solution. The amplitude of thechannel (10) gating will therefore reflect the analyte (20)concentration.

EXAMPLE

In order that the nature of the present invention may be more clearlyunderstood a preferred form thereof will now be described with referenceto the following example.

EXAMPLE Preparation of gramicidinN-epsilon-(2,4-dinitrophenyl)-N-alpha-(N-biotinyl)-6-amino-caproyl)lysineester (GaKDXB)

A mixture of gramicidin D (Sigma, 105 mg),N-alpha-BOC-N-epsilon-benzyloxycarbonyllysine (207 mg),dicyclohexylcarbodiimide (127 mg) and 4-(N,N-dimethylamino)pyridine (11mg) in dry dichloromethane (30 ml) was heated under reflux for 2 hoursthen stirred at room temperature overnight. The mixture was thenfiltered, evaporated to dryness and chromatographed on a silica gelcolumn eluted with dichloromethane/methanol/water/acetic acid(400:50:4:1) thence (400:60:6). The Ehrlich's reagent active fractionswere combined and evaporated to dryness. The residue was dissolved inmixture of dichloromethane (5 ml), methanol (5 ml), water (0.1 ml) andacetic acid (0.1 ml) and stirred with 10% palladium on charcoal (100 mg)under an atmosphere of hydrogen for 3 days. The mixture was filtered,evaporated no dryness and chromatographed on a silica gel column elutedwith dichloromethane/methanol/water (400:50:4) thence (400:60:6) toafford a polar, Ehrlich's agent reactive fraction of gramicidinN-alpha-BOC-lysine ester (78mg).

A solution of gramicidin N-alpha-BOClysine ester (78 mg) in a mixture ofdichloromethane (4 ml) and methanol (2 ml) was treated with2,4-dinitrofluorobenzene (50 μl). The mixture was stirred for 2 hoursthen evaporated to dryness. The residue was chromatographed on silicagel eluted with methanol/dichloromethane (5:95, 150 ml) thencedichloromethane/methanol/water (400/40/4), (200 ml) the front-runningyellow fractions which were eluted with dichloromethane/methanol/waterwere combined and evaporated to dryness to afford gramicidinN-alpha-BOC-N-epsilon-2,4-dinitrophenyllysine ester (34 mg).

A solution of gramicidin N-alpha-BOC-N-epsilon-2,4-dinitrophenyl lysineester (18 mg) was dissolved in trifluoroacetic acid (3 ml) and stirredfor 3 minutes then the solution was evaporated to dryness. The residuewas layered with toluene (5 ml) and evaporated to dryness (process wasrepeated 3 times). The residue was taken up in a mixture ofdichloromethane (4 ml) and methanol (2 ml) and the mixture was adjustedto pH 9 with triethylamine. N-Biotinyl-6-aminocaproic acidN-hydroxysuccinimide ester (10 mg) was then added to the solution andthe mixture was stirred at room temperature overnight. The mixture wasthen evaporated to dryness then chromatographed on a silica columneluted with dichloromethane/methanol (95:5, 150 ml) thendichloromethane/methanol/water (400:40:4,200 ml). The first three(yellow) fractions eluted with dichloromethane/methanol/water solventmixture were combined and evaporated to dryness to afford a yellowpowder of gramicidinN-epsilon-2,4-dinitrophenyl-N-alpha(N-biotinyl-6-amino-caproyl)lysineester (34 mg).

Construction of Membrane

Over a freshly evaporated gold electrode (2 mm² area) on a glasssubstrate was placed an open-ended cylindrical teflon sleeve (4 mm indiameter, 10 mm high). The teflon sleeve was secured in position by ametal clamp to create a tight seal at the glass-teflon interface. Asolution of glycerol monooleate (140 mM), reservoir lipid A(23-(20'-Oxo-19'-oxaeicosa-(Z)-9'-ene)-70-phenyl-20,25,28,42,45-pentaoxo-24-aza-19,29,32,35,38,41,46,47,52,55-decaoxa-58,59-dithiahexaconta-(Z)-9-ene, described in detail inPCT/AU93/00509) (140 μM) and gramicidin B (shown in FIG. 5 and describedin greater detail in PCT/AU93/00509) (1.4 μM) in a mixture oftetradecane and ethanol (1:9) (2 μl) was placed in the teflon wellassembly, followed immediately by a solution of sodium chloride (0.1M,100 μl). The assembly was then allowed to stand overnight. The aqueoussolution was then removed by syringe and the well was washed with water(100 μl) thence ethanol (3×100 μl). The assembly was allowed to dry thena solution of glycerolmonooleate (140 mM) and gramicidinN-epsilon-2,4-dinitrophenyl-N-alpha-(N-biotinyl-6-amino-caproyl)lysineester (14 μM) in a mixture of tetradecane and ethanol (1:9, 5 μl) wasadded. A solution of sodium chloride (0.1M, 100 μl) was immediatelyadded to the well and the well was then purged with 5×100 μl volumes of0.1M sodium chloride. The membrane assembly was then allowed to standunder a 100 μl volume of 0.1M sodium chloride overnight.

Assay of 2,4-dinitroaniline

A commercial preparation of rabbit anti-dinitrophenyl (DNP) antibody(Dakopatt, 7 mg/ml total antisera) was diluted 1:10 into 0.1M sodiumchloride solution. A series of samples were then prepared containing 90μl of the 1:10 dilution of anti-DNP antibody and 10 μl of a solution of2,4-dinitroaniline in water which had been acidified to pH 4.5 with HCl.The final concentration of 2,4-dinitroaniline in the solution rangedfrom 25 μM to 1 μM in twofold serial dilutions. The solutions wereallowed to stand overnight.

Sets of three sensor membranes for each 2,4-dinitroanilineconcentration, prepared as described above and monitored by AC impedancespectroscopy, were treated with a 2,4-dinitroaniline-antiDNP antibodysolution (5 μl). After 10 minutes a solution of streptavidin (0.5 mg/ml,2 μl) was added. The ratio of the AC impedance values immediately beforeand 5 minutes after streptavidin addition at the frequency giving thelargest impedance response to streptavidin was proportional to theamount of 2,4-dinitroaniline present in the initial sample added to themembrane (FIG. 4).

It is disclosed in co-pending International Patent Application No.PCT/AU93/00509 that advantages may be achieved by the exclusion oftetradecane in the preparation of membrane bilayers. It is not clear atthis point, however, whether any advantage will be gained in the presentsystem by the exclusion of tetradecane from the membrane solutions.

As will be readily understood by persons skilled in the art the firstand second ligands may be any of a large number of such bindingmolecules well known in the art. There is, however, the requirement thatthe second ligand is not so large that it prevents the binding of thefirst ligand to its binding partner even in the absence of the secondligand binding to its binding partner. Of course, in such a situation,there would be no meaningful gating of the ion channels.

As stated above the gating mechanism of the present invention is notrestricted to inhibition of streptavidin-biotinylated gramicidin gating.Any gating process which can be interfered with sterically will beamenable to this approach. For example it could be envisaged that thegating of gramicidin ion channels could be effected by the cross-linkingof adjacent gramicidin ion channels via the first ligand. In thisexample the first ligand could include a group which under oxidativeconditions could cross-link with a corresponding group on an adjacentgramicidin residue thereby preventing the ions across the membrane viathe gramicidin ion channels. The binding of the second ligand to itsbinding partner could then prevent the cross-linking and concomitantgating and, by analogy, to the biotin-streptavidin example above,provide another general detection scheme.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

I claim:
 1. A membrane for use in the detection of an analyte, themembrane comprising a closely packed array of self-assemblingamphiphilic molecules, a plurality of ionophores and a first and asecond ligand attached to an end of the ionophore adjacent the surfaceof the membrane, wherein the binding of the first ligand to its specificbinding partner prevents the flow of ions across the membrane via theionophores and wherein binding of the second ligand to its specificbinding partner prevents the binding of the first ligand to its specificbinding partner.
 2. A membrane as claimed in claim 1 in which the firstligand is biotin.
 3. A membrane as claimed in claim 1 in which theionophores are gramicidin or gramicidin analogues.
 4. A membrane asclaimed in claim 2 in which the ionophores are gramicidin or gramicidinanalogs.